The role of Grp75 in supercomplex assembly and neurodegeneration

越来越多的证据支持线粒体呼吸链(MRC)通过 被称为超复合体的有组织的多复合体结构。然而，动态和监管 超复杂组装还没有得到充分的研究。特别是，几乎没有任何调节蛋白质的因素 参与超复杂组装的分子已被确定。我们的长期目标是了解 线粒体呼吸机械及其潜在的调控机制。这一特殊情况的目标是 应用是确定线粒体伴侣，75 kDa葡萄糖调节蛋白(Grp75)是否发挥作用 在调节超复合体组装并进一步确定参与这一重要的额外蛋白质因子 进程。自从普通酵母以来，哺乳动物呼吸超复合体组装的研究一直是一个难点 可以作为一个强大的遗传学系统来识别假定的调控因素的系统，缺乏 复合体I是哺乳动物超复合体的重要组成部分。我们之前已经建立了一个 分离携带线粒体DNA(MtDNA)突变的细胞的有效方法 具有受调节/改变的超复合体组装的细胞模型，可能是由于增强/稳定 超络合物与调节因子的相互作用(S)。这些细胞系的特征采用 分子和蛋白质组学方法都涉及分子伴侣Grp75超复合体 集合。有趣的GRP75此前曾因1)而与帕金森氏病(PD)有关。GRP 75 已在PD患者中发现突变；2)PD患者脑组织中GRP75低表达；3) 我们的初步研究表明，杂合子Grp75小鼠表现出较低的运动活动，与 有缺陷的超复杂组件。这个应用的中心假设是Grp75是一个必不可少的部分 关于调节超复合体组装的机器，以及有缺陷的超复合体组装 与GRP75缺陷相关的会导致神经变性。为了检验这一假设，我们建议 追求以下三个具体目标：1)确定Grp75在超复合体组装中的作用。在……里面 特别是，我们将跟踪单个络合物和超络合物的逐步组装和降解 在有或没有Grp75和实验室新开发的方法的情况下；2)确定 Grp75调控超复合体的新蛋白因子及其作用机制 集合。用蛋白质组学方法分析Grp75相互作用的蛋白质和含有 在具有调节/改变的超复合体组装的细胞模型中，我们的目标是分离新的 参与调节超复合体组装的蛋白质因素。3)用改变的小鼠模型来表征 Grp75的表达。有缺陷的超复合体动力学在神经元变性中的意义将是 进一步探索杂合子和神经元特异性Grp75基因敲除小鼠模型。我们将调查 Grp75缺陷到超复合体缺陷再到神经元的潜在分子通路 退化。这种方法是创新的，因为它结合了我们独特的细胞模型，展示了 超复杂动力学与新开发的分析方法，使我们能够理解 呼吸系统超复合体组装。新型缺陷超复合体小鼠模型的建立 动力学应该为研究神经系统和神经退行性变的生物能量学提供新的可能性。 我们相信，我们在描述呼吸道超复合体组装方面处于有利地位。这项研究 具有重要意义，因为阐明这一机制可以为氧化调节提供新的见解 磷酸化机器。此外，我们预计我们的工作也将有助于识别新的风险基因。 参与与线粒体功能障碍相关的神经退行性疾病。

Emerging evidence supports the proposition that the mitochondrial respiratory chain (MRC) functions via organized multicomplex structures called supercomplexes. However the dynamics and regulation of supercomplex assembly have not been fully investigated. In particular, hardly any regulatory protein factors involved in supercomplex assembly have been identified. Our long term goal is to understand the dynamics of mitochondrial respiratory machinery and its underling regulatory mechanism. The objective of this particular application is to determine if the mitochondrial chaperon, 75 kDa glucose regulated protein (Grp75) plays a role in regulating supercomplex assembly and further to identify additional protein factors involved in this important process. The study of mammalian respiratory supercomplex assembly has been difficult since common yeast systems, which could be utilized as a powerful genetics system to identify putative regulatory factors, lack Complex I an essential component of mammalian supercomplexes. We have previously established an efficient method to isolate cells carrying mitochondrial DNA (mtDNA) mutations and further generated several cell models with regulated/altered supercomplex assembly, probably due to the enhanced/stabilized interactions between supercomplexes and regulative factor(s). Characterizations of these cell lines employing both molecular and proteomics approaches have implicated the molecular chaperone Grp75 supercomplex assembly. Interesting Grp 75 has previous been implicated in Parkinson's diseases (PD) due to 1). Grp 75 mutations have been identified in PD patients; 2) Low Grp 75 expression was found in brains of PD patients; 3). Our preliminary studies showed heterozygous Grp75 mice exhibited lower motor activities associated with defective supercomplex assembly. The central hypothesis for this application is that Grp75 is an essential part of machinery which regulates the assembly of supercomplexes, and defective of supercomplex assembly associated with deficient Grp 75 would lead to neuro-degeneration. To test this hypothesis, we propose to pursue the following three specific aims: 1) Characterize the role of Grp75 in supercomplex assembly. In particular, we will follow the step-wise assembly and degradation of individual complexes and supercomplexes in presence and absence of Grp75 with newly developed approaches in the lab; 2) Determine the regulatory mechanisms of Grp75 on supercomplex by Identify novel protein factors involved in regulating supercomplex assembly. With proteomic analysis of proteins interacting with Grp75 and Complex I containing supercomplexes in the cell models with regulated/altered supercomplex assembly, we aim to isolate novel protein factors involved in regulating supercomplex assembly. 3) Characterize the mouse models with altered expression of Grp75. The implications of defective supercomplex dynamics in neuronal degeneration will be further explored in heterozygous and neuronal-specific Grp75 knockout mouse models. We will investigate the underlying molecular pathways derived from Grp75 defect to supercomplex deficiency to neuronal degeneration. The approach is innovative, because it combines our unique cell models exhibiting upregulated supercomplex dynamics with newly-developed analytical methods to allow understanding of the complexity of respiratory supercomplex assembly. The establishment of novel mouse models with defective supercomplex dynamics should open new possibilities to study bioenergetics in neuronal system and neuro-degeneration. We believe that we are in a strong position to characterize respiratory supercomplex assembly. The research is significant, because elucidating this mechanism could provide new insights into the regulation of oxidative phosphorylation machinery. In addition, we anticipate our work will also help to identify novel risk genes involved in neurodegenerative diseases associated with mitochondrial dysfunction.